Information on EC 2.7.4.16 - thiamine-phosphate kinase

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The expected taxonomic range for this enzyme is: Bacteria, Archaea

EC NUMBER
COMMENTARY hide
2.7.4.16
-
RECOMMENDED NAME
GeneOntology No.
thiamine-phosphate kinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + thiamine phosphate = ADP + thiamine diphosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
thiamine diphosphate biosynthesis I (E. coli)
-
-
thiamine diphosphate biosynthesis II (Bacillus)
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-
Thiamine metabolism
-
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thiamine salvage I
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thiamine salvage II
-
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vitamin B1 metabolism
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-
SYSTEMATIC NAME
IUBMB Comments
ATP:thiamine-phosphate phosphotransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
9068-23-9
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
Uniprot
Manually annotated by BRENDA team
K12
-
-
Manually annotated by BRENDA team
gene Pcal_0657 or THI80
UniProt
Manually annotated by BRENDA team
gene Pcal_0657 or THI80
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ADP + thiamine phosphate
?
show the reaction diagram
ATP + thiamine phosphate
ADP + thiamine diphosphate
show the reaction diagram
ITP + thiamine phosphate
?
show the reaction diagram
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + thiamine phosphate
ADP + thiamine diphosphate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
requirement, about 95% as effective as Mg2+
Fe2+
-
activation, about 10% as effective as Mg2+
K+
-
activation
Mn2+
-
activation, about 30% as effective as Mg2+
NH4+
-
activation
Rb+
-
activation
Zn2+
-
activation, about 30% as effective as Mg2+
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ADP
-
product inhibition
Cs+
-
antagonizes stimulation by K+
diphosphate
-
-
Li+
-
antagonizes stimulation by K+
N-ethylmaleimide
-
-
Na+
-
antagonizes stimulation by K+
Oxythiamine
-
-
PCMB
-
2-mercaptoethanol reverses
pyrithiamine
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-
Pyrithiamine phosphate
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-
thiamine
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-
additional information
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no inhibition by phosphate, KCN, iodoacetic acid, arsenate or arsenite
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00013 - 0.28
ATP
0.000043 - 0.0013
thiamine phosphate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.006 - 0.092
thiamine phosphate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.33 - 46
ATP
4
68 - 320
thiamine phosphate
4570
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.53 - 0.56
AMP
0.36
pyrithiamine
-
pH 7.5, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000197
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-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80 - 120
10% of maximal activity at 80C, 50% at 100C, maximal activity at 120C, inactive on ice
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Methylobacillus flagellatus (strain KT / ATCC 51484 / DSM 6875)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35000
recombinant enzyme, amino acid sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme in complex with nonhydrolyzable AMP-PCP and TMP, or with the reaction products ADP and TPP, ThiL is incubated with 4 mM AMP-PCP or ATP and 4 mM MgCl2 on ice for 1 h prior to crystallization experiments, optimized conditions for the ThiL binary complexes include 100 mM HEPES, pH 7.4, 6-11% isopropyl alcohol, 200 mM NaCl, and 4-6% 2-methyl-2,4-pentanediol, 100 mM CaCl2 is used as an additive in a 1:9 ratio with the reservoir solution, TMP is soaked into the co-crystallized ThiL and AMP-PCP crystals by transferring the crystals to a solution containing the mother liquor and 20 mM TMP and allowing the crystals to soak for 5 min before being frozen. To obtain ThiL ADP-TPP crystals, ThiL is incubated on ice with 4 mM ADP, 4 mM TPP, and 4 mM MgCl2, optimized conditions for the product complex include 100 mM imidazole, pH 8.0, 7-10% PEG 8000, and 100-250 mM Ca(C2H3O2)2, 3-7 days, cryoprotection by 15% ethylene glycol, X-ray diffraction structure determination and analysis at 1.5-2.6 A resolution
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
100
purified recombinant ThiL protein, loss of about 45% activity after 15 min at 100C and 20% after 60 min, ATP can partially stabilize the activity, addition of 0.5 M NaCl or 0.5 M KCl to the enzyme solution does not improve the thermostability
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
ATP stabilizes the enzyme
glycerol stabilizes during purification
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 3 days, t1/2: 10 days
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by metal affinity chromatography and ultrafiltration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construction of the thiamine monophosphate kinase (thiL) overexpression plasmid, overexpression from pCLK710
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encoded by thiL, thiL is not transcriptionally regulated by thiamine or thiamine diphosphate
expression in Escherichia coli
gene THI80 or Pcal_0657, sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R136M
site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type
S196A
site-directed mutagenesis, the mutant shows 90% reduced activity compared to the wild-type
R136M
-
site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type
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S196A
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site-directed mutagenesis, the mutant shows 90% reduced activity compared to the wild-type
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